Differential TIMP3 expression affects tumor progression and angiogenesis in melanomas through regulation of directionally persistent endothelial cell migration
Journal: Angiogenesis
Asha M. Das1 (MSc), Ann L.B. Seynhaeve1 (PhD), Joost A.P. Rens1 (BSc), Cindy E. Vermeulen1 (BSc), Gerben A. Koning1 (PhD), Alexander M.M. Eggermont1,2 (MD, PhD), and Timo L.M. ten Hagen1 (PhD)
1Laboratory Experimental Surgical Oncology, Section Surgical Oncology, Department of Surgery, Erasmus Medical Center, The Netherlands
2Institut de Cancérologie Gustave Roussy, Villejuif, Paris, France
Running title: TIMP3 and angiogenic potential of melanomas
Supplementary Information: 1 document, 5 figures and 7 videos
Correspondence: Dr. Timo L.M. ten Hagen, Laboratory Experimental Surgical Oncology, Section Surgical Oncology, Department of Surgery, Erasmus MC, Room Ee 0104a, PO Box 1738, 3000 DR Rotterdam, The Netherlands, tel. +31 (0)10 70 43682 / 44568, fax. +31 (0)10 70 44746, email:
Supplemental Information
Materials and methods
Reagents
Tissue culture reagents, unless otherwise specified, were from Biowhittaker (Walkersville, MD). Human recombinant basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were from PeproTech (Rocky Hill, NJ). Fibronectin was from Roche Diagnostics Nederland B.V. (Almere, NL). Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) reagents were from BioRad (Hercules, CA). Rabbit anti-human TIMP3-Loop 1 polyclonal antibody and mouse anti-human beta actin monoclonal antibody were from Abcam (Cambridge, UK). TRIzol reagent, First Strand cDNA synthesis kit and Platinum SYBR Green qPCR Super-Mix-UDG were from Invitrogen (Carlsbad, CA). All other reagents were from Sigma-Aldrich Chemie B.V. (Zwijndrecht, NL), unless otherwise stated.
Cell culture
Primary EC cultures from two separate donors were used for this study. Primary EC cultures were established using collagenase digestion as previously described [1]. EC’s were routinely cultured in Human Endothelial-SFM (Invitrogen) supplemented with 20% heat inactivated new born calf serum, 10% heat inactivated human serum, 100 ng/mL bFGF and 100 ng/mL EGF, on 0.1% gelatin coated flasks. All migration assays were performed with ECs between passage 3 and 5. The human melanoma cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with glutamine supplemented with 10% fetal calf serum (FCS). ECs and tumor cells were routinely cultured in a well humified incubator (37°C in 5% CO2, 20% O2), and subcultured when confluent.
Real-time RT-PCR
Differential expression of TIMPs in the melanoma cell lines was verified with qPCR. Total RNA extraction was performed with TRIzol reagent, according to the manufacturer’s instructions. Reverse transcription was performed with 2 µg total RNA using the First-Strand cDNA Synthesis Kit. 200 ng of cDNA was used for the quantitative PCR (qPCR) reaction. qPCR was performed in triplicates, using the iCycler (Bio-Rad Laboratories, Munich, Germany) with Platinum SYBR Green qPCR Super-Mix-UDG. Specific PCR primer pairs were synthesized and obtained from Invitrogen. The sequences of the PCR primer pairs used are as follows:
TIMP1 (GenBank RefSeq NM_003254): Forward: 5’-GACGGCCTTCTGCAATTCC-3’, Reverse: 5’-GTATTAGGTGGTCTGGTTGACTTCTG-3’
TIMP2 (GenBank RefSeq NM_003255): Forward: 5’-GAGCCTGAACCACAGGTACCA-3’, Reverse: 5’-TCTGTGACCCAGTCCATCCA-3’
TIMP3 (GenBank RefSeq NM_000362): Forward: 5’-CCAGGACGCCTTCTGCAA-3’, Reverse: 5’-CCCCTCCTTTACCAGCTTCTTC-3’
TIMP4 (GenBank RefSeq NM_003256): Forward: 5’-CCAGAGGTCAGGTGGTAA-3’, Reverse: 5’-ACAGCCAGAAGCAGTATC-3’
HPRT (GenBank RefSeq NM_ 000194): Forward: 5’-TATTGTAATGACCAGTCAACAG-3’, Reverse: 5’-GGTCCTTTTCACCAGCAAG-3’
The reactions were incubated in a 96-well optical plate at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 30 s. Post qPCR, a melting curve analysis was performed. Gene expression values were normalized to the housekeeping gene HPRT. Differences in transcript abundance are plotted as log2 ratio relative to a selected mean expression. Specific PCR products were additionally visualized on 2% agarose gels (TIMP1: 79 BP, TIMP2: 114 bp, TIMP3: 73 bp, HPRT: 192 bp).
Preparation of cell lysates and Western Blot analysis
TIMP3 protein levels in CM and melanoma cells were detected using SDS-PAGE. Cells were washed in cold PBS and lysed in ice-cold lysis buffer (50mM Tris-HCL pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and proteinase inhibitor cocktail) for 30 min on ice. Lysates were cleared from cellular debris by centrifugation for 10 min at 12000g, 4°C. Protein concentration in cell lysates and CM was measured with Coomassie Plus Reagent (Pierce, Rockford, IL). Samples were normalized for protein content, electrophoresed on 12% gels, and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1 h at room temperature with 5% nonfat dried milk in PBS/0,05% Tween-20, followed by incubation with an antibody to TIMP3-Loop 1 (0.1 µg/ml) and beta actin (1:5000) diluted in 5% nonfat dried milk in PBS/0,05% Tween-20 overnight at 4ºC. Post washing, the membranes were incubated with IRDYE labeled secondary antibody (LI-COR) for 1 h at room temperature and scanned using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).
Reverse zymography
To assay for metalloproteinase inhibitory activity of TIMP-3 in melanoma CM samples, reverse zymography was performed using protease-substrate gel electrophoresis. After mixing the samples with Laemmli sample buffer (Bio-Rad), 6 µl of the sample (without heating or reducing agents) was separated by electrophoresis in a matrix consisting of 0.1% SDS, 12% polyacrylamide gel containing 250 ng/ml active gelatinase A (Calbiochem, Cambridge, MA) and 2.5 mg/ml gelatin. After electrophoresis, gels were washed for 3 h in 2.5% Triton X-100 to remove the SDS and restore enzyme activity for incubation. Gels were incubated for 16 h at 37°C in of 50 mM Tris–HCl, pH 7.6, 0.2 M NaCl, 5 mM CaCl2, 0.02% Brij-35 and 0.02% sodium azide. Finally, gels were stained for 4 h in 2.5 mg/ml Coomassie G-250 in 30% methanol, 10% acetic acid and then destained in 30% methanol, 10% acetic acid. Presence of active TIMP2 and TIMP3 was visible as dark stained bands against a clear background. Band intensities were quantified using NIH Image J software and were normalized to melanoma cell numbers used to generate the respective melanoma CMs.
TIMP3 shRNA transfection
For stable TIMP3 silencing, five different shRNA vectors with neomycin resistance gene were used (four target specific shRNAs targeting human TIMP3 and one negative control shRNA vector) (SABiosciences, Fredrick, MD). The plasmids were used to transform E.coli and the product was amplified. For transfections, 1F6 melanoma cells were seeded in a 6-well plate at a concentration of 1×106 cells/well and allowed overnight growth to reach 60-70% confluency. Cells were then transfected with a mixture of 1 μg plasmid DNA and 2.5 μl of LipofectAMINE 2000 (Invitrogen) in 1 ml OptiMEM-1 medium (Invitrogen). At 24 h post-transfection, the medium was replaced by normal medium (containing 10% FCS) and cultured up to 48 h. Stable clones were isolated and propagated using neomycin selection (selection medium containing 10% FCS and 0.75 mg/ml G418). Gene knockdown was determined with qPCR.
MMP inhibition
MMP inhibition was achieved by adding the synthetic MMP inhibitor, GM6001 (10 µM) to the melanoma CM. As a control, GM6001 Negative Control (10 µM) was used.
Cell proliferation assay
To assess the effect of TIMP3 on the proliferation of melanoma cells, cells were seeded at a density of 1 X 104 cells/ well (for modified and unmodified 1F6 cells) and 6 X 103 cells per well (for modified and unmodified BLM cells) in 96-well cluster plates and allowed to grow up to 96 h. At 24, 48, 72 and 96 h, cells were fixed with 10% trichloroacetic acid, washed under tap water and stained with Sulforhodamine B (SRB). After washing with 1% acetic acid, plates were dried at 50°C and the dye was solubilized in 10mM Tris buffer (all Sigma-Aldrich). The absorbance was measured using a microplate reader (Victor 1420, Wallac, Turku, Finland) at 510 nm. All proliferation data were normalized to the 24 h time point, and is expressed as a percentage.
Alternatively, to evaluate the effect of TIMP3 in the melanoma CM on the proliferation of ECs, ECs were seeded at a density of 6 X 103 cells per well in 96-well cluster plates and grown to 60% confluence. After 24 h in endothelial culture medium, CM from modified and unmodified melanoma cells (supplemented with or without FCS) was added to ECs, and incubations continued for 24-72 h. After 24-72 h, cells were fixed with 10% trichloroacetic acid, washed under tap water and stained with Sulforhodamine B (SRB). After washing with 1% acetic acid, plates were dried at 50°C and the dye was solubilized in 10mM Tris buffer (all Sigma-Aldrich). The absorbance was measured using a microplate reader (Victor 1420, Wallac) at 510 nm. All EC proliferation data were normalized to the 0 h time point (time of addition of melanoma CMs), and is expressed as a percentage.
Cell adhesion assay
To assess the effect of the various melanoma CMs on the adhesive capacity of ECs, ECs were incubated in suspension with CMs at a concentration of 15*104 cells/ mL. After incubation for 4 h at 37°C, ECs were seeded at a density of 15*103 per well in 96 well cluster plates. Cells were allowed to adhere up to 1 h. At each time point, non-adherent cells were washed off and adherent cells were fixed with 10% trichloroacetic acid and stained with Sulforhodamine B (SRB). After washing with 1% acetic acid, plates were dried at 50°C and the dye was solubilized in 10mM Tris buffer (Sigma-Aldrich). The absorbance was measured using a microplate reader (Victor 1420, Wallac) at 510 nm.
3D dispersion assay
The effect of TIMP3 in the melanoma CM on the invasive capacity of ECs was determined using a cell dispersion assay with Cytodex-3 microcarrier beads (Sigma-Aldrich). The microcarrier beads were prepared under sterile conditions, according to the manufacturer’s instructions. ECs were mixed with the sterile microcarrier beads in a falcon tube considering a ratio of 25 cells per bead. The suspension was incubated at 37°C for 24h, with gentle mixing every two hours to ensure complete coating of the beads. Next, the suspension was transferred to a T25 cm2 culture flask to remove unattached cells and the cells were allowed to grow on the beads for another 2 days. The coated microcarrier beads were embedded in 2 mg/ml collagen gel (collagen: MEM: NaHCO3 in the ratio 8:1:1) in a 24 well cluster plate, such that each well had 150 beads. The plates were incubated at 37°C for 2h for the beads to settle in the gel and the polymerized gels were covered with 500 uL of 2x concentrated melanoma CM treatments, supplemented with 20% FCS. Cell dispersion was followed for up to 96 h with a 10X/0.30 PLAN-NEOFLUAR objective lens (Carl Zeiss).
Migration assay with subconfluent ECs
Migration assays with subconfluent cultures of ECs were peformed by seeding 5*104 ECs in a sterile, fibronectin coated migration ring, followed by incubation at 37°C. After 24 h, the cells were washed twice, followed by the addition of treatments and migration assays were conducted as described earlier. Quantifications were performed on 10 randomly chosen cells, per experiment, in the migration field. At least four experiments were performed with the treatments tested.
Results
TIMP3 in melanoma conditioned-medium inhibits endothelial cell migration, at least in part, via MMP-independent pathways
To investigate whether TIMP3 mediated inhibitory effects on ECs via MMP-dependent or MMP-independent pathways, we performed migrations with BLM-EV and BLM-TIMP3 CM in the presence of the broad spectrum MMP inhibitor, GM6001 or the mock inhibitor analogue, GM6001 Negative Control. We observed a reduction in both the total and effective migration induced by BLM-EV CM in the presence of GM6001 when compared to the negative control (Supplementary Fig. 1a, b, c; P<.005 and P<.05 respectively). Interestingly, treatment with BLM-TIMP3 CM, in the presence of GM6001 or negative control did not cause significant differences in the total or effective migrated distances (Supplementary Fig. 1a, b, c; ns). Moreover, treatment with BLM-TIMP3 CM, in the presence of both GM6001 and the negative control resulted in lower effective migration as compared to BLM-EV treatment with GM6001. These data suggest that TIMP3 inhibits EC migration, at least in part, via MMP independent pathways. A previous study has reported the inhibition of VEGF induced chemotactic migration of ECs upon addition of recombinant human TIMP3 (rhTIMP3) by engagement of the VEGF receptor 2 [2].
TIMP3 in melanoma conditioned-medium does not affect endothelial cell proliferation
To evaluate if the TIMP3 in the melanoma conditioned-medium had an effect on EC proliferation, we treated ECs with CMs from modified and unmodified melanoma cells in the absence or presence of 10% FCS. Control treatments were performed by supplementing basal medium with rhTIMP3. We did not observe a significant difference in EC proliferation when treated with CMs of BLM, BLM-EV or BLM-TIMP3 cells, in the absence or presence of FCS (Supplementary Fig. 2a). Similarly, no significant difference in EC proliferation was observed when treated with the CMs of 1F6, 1F6 shTIMP3 or 1F6 shControl cells, in the absence or presence of FCS (Supplementary Fig. 2b). Moreover, treatment with rhTIMP3 did not affect EC proliferation (Supplementary Fig. 2c). Our data is in accordance with previously published reports of the lack of effect of TIMP3 on the proliferative capacity of ECs [3].
TIMP3 in melanoma conditioned-medium increases endothelial cell adhesion
Studies on normal [4] and tumor [5] cells have demonstrated the effect of TIMP3 in the modulation of cell adhesion. To assess if TIMP3 had an effect on the adhesive property of ECs, we performed an adhesion assay where ECs were pretreated with the different melanoma CMs for 4 h and then plated in the presence of the same media, in non-coated or fibronectin coated 96 well cluster plates. We measured the number of attached cells at 5, 10, 15, 30 and 60 mins after seeding. As shown in Supplementary Fig. 2d and e, we observe that treatment with BLM-TIMP3 CM or BLM-EV CM supplemented with rhTIMP3 (2 ng/mL, concentration of TIMP3 in BLM EV CM as measured by ELISA) results in more ECs adhering to both substrates as compared to ECs treated with BLM and BLM-EV CM, at all time points. Likewise, treatment with 1F6 and 1F6 shControl CM resulted in increased adhesion of ECs to the substrate when compared to 1F6 shTIMP3 CM. ECs treated with 1F6 shTIMP3 CM in the presence of 3 ng/mL rhTIMP3 (concentration of TIMP3 in 1F6 shControl CM as measured by ELISA) resulted in increased cell adhesion (Supplementary Fig. 2f and g). It is however important to note that the differences in EC adhesion with the different 1F6 treatments were more visible at the earlier time points. Taken together, these data indicate that TIMP3 is involved in cell-substrate adhesion in a quantitative manner and might, in extension, modulate cell-ECM interactions and migration.